The research project concerns the mechanism by which the IkappaB-alpha inhibitor becomes inactivated during stimulation of immune cells which in turn leads to activation of the NF-kappaB transcription factor. NF- kappaB is critical for the induced expression of many genes whose encoded functions are required to counteract pathogens or stress. In addition, NF-kappaB regulates the expression of several viruses, including the human immunodeficiency virus (HIV). Therefore, blocking the activation of NF-kappaB could aid in the treatment of many inflammatory diseases and may also prevent the spread of HIV. An understanding of the mechanism(s) of activation of this transcription factor and the identification of the molecular components involved will provide potential targets for anti-inflammatory and anti-viral therapies. We have previously demonstrated that activation of the NF-kappaB transcription factor involves rapid phosphorylation and proteolytic degradation of its cytoplasmic inhibitor IkappaB-alpha. We discovered that the phosphorylation of this inhibitor in response to cellular stimulation is not sufficient to activate the transcription factor, but that induced proteolytic degradation is necessary. Specifically we found that calpain inhibitors will block activation of the transcription factor by blocking the proteasome-mediated degradation of IkappaB-alpha, while having no effect on its induced phosphorylation. We determined the precise amino acid sites at which the inhibitor becomes phosphorylated in response to signals, and we showed that phosphorylation of these sites is required for proteolytic degradation and thus for activation of NF- kappaB. These results will aid in the identification of the molecular component(s) which targets the phosphorylated inhibitor for proteasomal degradation and the kinase(s) which phosphorylates in response to appropriate stimuli.